Over-expression of STP13, a hexose transporter, improves plant growth and nitrogen use in Arabidopsis thaliana seedlings.

In Arabidopsis thaliana, the regulation of hexose levels by the large monosaccharide transporter (MST) gene family influences many aspects of plant growth. The cloning and transgenic expression of one family member (STP13) enabled the manipulation of carbon (C) and nitrogen (N) metabolism in Arabidopsis. Transgenic seedlings constitutively over-expressing STP13 (STP13OX) had increased rates of glucose uptake, higher endogenous sucrose levels and accumulated more total C and biomass per plant when grown on soil-less media supplemented with 55 mM glucose and sufficient N (9 mM nitrate). Furthermore, STP13OX seedlings acquired 90% more total N than the Col-0 seedlings, and had higher levels of expression of the nitrate transporter NRT2.2. In addition, STP13OX seedlings were larger and had higher biomass than Col-0 seedlings when grown under a limiting N condition (3 mM nitrate). Transgene analysis of STP13 reveals that its gene product is localized to the plasma membrane (PM) in tobacco BY-2 suspension cells, that it encodes a functional MST in planta, and that the STP13 promoter directs GUS expression to the vasculature and to leaf mesophyll cells. This work highlights the link between C and N metabolism, demonstrating that a plant's N use may be improved by increasing the availability of C.

A novel strategy for regulated expression of a cytotoxic gene.

The tetracycline (Tet) transactivator system is a powerful promoter system to control gene expression. However, expression of a cytotoxic gene in this system has been limited due to the lethal effect caused by low levels of basal expression of the toxic gene. In this report, we describe a novel strategy to express a toxic gene using the Tet system. The barstar gene is placed downstream of a minimal promoter and the barnase gene downstream of the tetracycline responsive element minimal promoter. When barnase is expressed at a basal level, its toxicity in human cell culture is offset by the similar basal level expression of barstar. However, when the barnase expression is induced with the transactivator protein, its overproduction leads to cell death. Therefore, this strategy allows cytotoxicity to be effectively regulated by tetracycline.

Self-aggregation characteristics of recombinantly expressed human elastin polypeptides.

Elastin is an extracellular matrix protein found in tissues requiring extensibility and elastic recoil. Monomeric elastin has the ability to aggregate into fibrillar structures in vitro, and has been suggested to participate in the organization of its own assembly into a polymeric matrix in vivo. Although hydrophobic sequences in elastin have been suggested to be involved in this process of self-organization, the contributions of specific hydrophobic and crosslinking domains to the propensity of elastin to self-assemble have received less attention. We have used a series of defined, recombinant human elastin polypeptides to investigate the factors contributing to elastin self-assembly. In general, coacervation temperature of these polypeptides, used as a measure of their propensity to self-assemble, was influenced both by salt concentration and polypeptide concentration. In addition, hydrophobic domains appeared to be essential for the ability of these polypeptides to self-assemble. However, neither overall molecular mass, number of hydrophobic domains nor general hydropathy of the polypeptides provided a complete explanation for differences in coacervation temperature, suggesting that the specific nature of the sequences of these hydrophobic domains are an important determinant of the ability of elastin polypeptides to self-assemble.

Expression of the S receptor kinase in self-compatible Brassica napus cv. Westar leads to the allele-specific rejection of self-incompatible Brassica napus pollen.

Expression of an S receptor kinase (SRK910) transgene in the self-compatible Brassica napus cv. Westar conferred on the transgenic pistil the ability to reject pollen from the self-incompatible Brassica napus W1 line, which carries the S910 allele. In one of the SRK transgenic lines, 1C, virtually no seeds were produced when the transgenic pistils were pollinated with W1 pollen (Mean number of seeds per pod = 1.22). This response was specific to the W1 pollen since pollen from a different self-incompatible Brassica napus line (T2) and self-pollinations were fully compatible. Westar plants expressing an S locus glycoprotein transgene (SLG910) did not show any self-incompatibility response towards W1 pollen. Transgenic Westar plants resulting from crosses between the 1C SRK transgenic line and three SLG910 transgenic lines were also tested for rejection of W1 pollen. The additional expression of the SLG910 transgene in the SRK910 transgenic plants did not cause any significant further reduction in seed production (Mean seeds/pod = 1.04) or have any detectable effects on the number of pollen grains that adhered to the pistil. Thus, while the allele-specific SLG gene was previously reported to have an enhancing effect on the self-incompatibility response, no evidence for such a role was found in this study.

Molecular mechanisms of self-recognition in Brassica self-incompatibility.

Plants have mechanisms to promote outbreeding and thereby to increase their genetic diversity. In species that are self-incompatible, self-pollen is rejected by the stigma. This mechanism has been the subject of intense study for many years and, in the past two years, significant progress has been made in identifying the genes involved in Brassica. Self-recognition involves two genes, one of which determines the male and the other the female specificity. Considerable progress has also been made on the mechanism by which self-recognition leads to pollen rejection, although the delineation of all the genes involved is still not complete.

Transformation of Arabidopsis with a Brassica SLG/SRK region and ARC1 gene is not sufficient to transfer the self-incompatibility phenotype.

Self-incompatibility (SI) promotes outbreeding in flowering plants, and in Brassica SI is genetically controlled by the S locus. Self-incompatible Brassica and self-fertile Arabidopsis belong to the same crucifer family. In addition, a comparative analysis reveals a high degree of microsynteny between the B. campestris S locus and its homologous region in Arabidopsis--with the notable exception that the Brassica SI genes, SLG and SRK, are missing. Brassica ARC1 encodes a component of the SRK signal transduction pathway leading to self-pollen rejection, and no closely related ARC1 homolog has been identified in Arabidopsis. The purpose of the research reported here was to introduce Brassica SI components into Arabidopsis in an attempt to compensate for the missing genes and to investigate whether the SI phenotype can be transferred. Inserts of approximately 40 kb from the fosmid clones F20 and F22, which span the B. napus W1 SLG-SRK region, were cloned into the plant transformation vector pBIBAC2. Transgenic plants were generated that expressed the Brassica SI genes in the flower buds. In addition, the endogenous, SLG-like, gene AtS1 was not co-suppressed by the Brassica SLG transgene. No SI phenotype was observed among the T1 BIBAC2-F20 and BIBAC2-F22 transgenic plants. When the ARC1 gene was transformed into BIBAC2-F20 or BIBAC2-F22 plants, the resulting BIBAC2-F20-ARC1 and BIBAC2-F22-ARC1 plants still set seeds normally, and no rejection response was observed when self-incompatible B. napus W1 pollen was placed on BIBAC2-F20-ARC1 or BIBAC2-F22-ARC1 Arabidopsis stigmas. Taken together, our results suggest that complementing Arabidopsis genome with Brassica SLG, SRK and ARC1 genes is unlikely to be sufficient to transfer the SI phenotype.

Expression of allene oxide synthase determines defense gene activation in tomato.

Allene oxide synthase (AOS; hydroperoxide dehydratase; EC 4.2.1.92) catalyzes the first step in the biosynthesis of jasmonic acid from lipoxygenase-derived hydroperoxides of free fatty acids. Using the AOS cDNA from tomato (Lycopersicon esculentum), in which the role of jasmonic acid in wound-induced defense gene activation has been best described, we examined the kinetics of AOS induction in response to wounding and elicitors, in parallel with that of the wound-inducible PIN II (proteinase inhibitor II) gene. AOS was induced in leaves by wounding, systemin, 12-oxophytodienoic acid, and methyl jasmonate. The levels of AOS mRNA started declining by 4 h after induction, whereas the levels of PIN II mRNA continued to increase up to 20 h after induction. Salicylic acid inhibited AOS and PIN II expression, and the addition of 12-oxophytodienoic acid or methyl jasmonate did not prevent the inhibition of PIN II expression in the presence of salicylic acid. Ethylene induced the expression of AOS, but the presence of ethylene alone did not produce an optimal induction of PIN II. The addition of silver thiosulfate, an ethylene action inhibitor, prevented the wound-induced expression of both AOS and PIN II. Products of hydroperoxide lyase affected neither AOS nor PIN II, but induced expression of prosystemin. Based on these results, we propose an updated model for defense gene activation in tomato.

A binary vector-based large insert library for Brassica napus and identification of clones linked to a fertility restorer locus for Ogura cytoplasmic male sterility (CMS).

We constructed and characterized a large DNA insert library for Brassica napus that would facilitate genome-related research and map-based cloning efforts in Brassica species. This library, consisting of 92,160 clones arrayed in 384-well microtiter dishes, was based on a conventional plant transformation vector (binary vector), and was constructed using a single ligation with transformation efficiency of over 5000 recombinants per microliter of ligation mixture. Every clone in this library contains an insert in the size range of 30-190 kb, facilitating both chromosome walking and plant transformation. Screening this library with three DNA markers (C2, F10, and CabR) that are linked to a fertility restorer locus for Ogura cytoplasmic male sterility (CMS) identified at least 17 positive clones for each probe. Among the 17 positive clones identified by C2, nine are linked to the restorer locus. Marker F10 identified 21 clones, of which only two are linked to the restorer locus. None of 68 clones identified by CabR is linked to the restorer locus. A stability test using two clones identified by the C2 marker indicated that large DNA inserts are stable in this conventional vector in both Escherichia coli and Agrobacterium.

The S locus glycoprotein and the S receptor kinase are sufficient for self-pollen rejection in Brassica.

Self-incompatibility (SI) is one of several mechanisms that have evolved to prevent inbreeding in plants. SI in Brassica is controlled by the polymorphic S locus complex. Two S locus-encoded proteins are coordinately expressed in the stigma epidermis: the cell wall-localized S locus glycoprotein (SLG) and the plasma membrane-anchored S receptor kinase (SRK). These proteins are thought to recognize a pollen factor that leads to the rejection of self-pollen. Evidence has accumulated that indicates that both proteins are necessary for the ability of the stigma to inhibit self-pollen. However, it has not been possible to prove this necessity definitively or to demonstrate that these genes are sufficient for this phenotype, because previous attempts to transfer this phenotype via transformation have not been successful. In this study, two overlapping S locus genomic clones, which cover approximately 55 kilobases of DNA and contain the SLG, SRK, and an anther-expressed gene in the region common to the two, were introduced into a self-compatible Brassica napus line. The resulting transgenic plants were shown to carry the female part of the SI phenotype, rejecting pollen in a haplotype-specific manner. However, the pollen SI phenotype was not found in any of the transgenic plants. These data show that the SLG and SRK are sufficient for the female side but not the male side of the SI phenotype in Brassica and that there must be an independent pollen S factor encoded outside the cloned region.

Structural and transcriptional comparative analysis of the S locus regions in two self-incompatible Brassica napus lines.

Self-incompatibility (SI) in Brassica is controlled by a single locus, termed the S locus. There is evidence that two of the S locus genes, SLG, which encodes a secreted glycoprotein, and SRK, which encodes a putative receptor kinase, are required for SI on the stigma side. The current model postulates that a pollen ligand recognizing the SLG/SRK receptors is encoded in the genomic region defined by the SLG and SRK genes. A fosmid contig of approximately 65 kb spanning the SLG-910 and SRK-910 genes was isolated from the Brassica napus W1 line. A new gene, SLL3, was identified using a novel approach combining cDNA subtraction and direct selection. This gene encodes a putative secreted small peptide and exists as multiple copies in the Brassica genome. Sequencing analysis of the 65-kb contig revealed seven additional genes and a transposon. None of these seven genes exhibited features expected of S genes on the pollen side. An approximately 88-kb contig of the A14 S region also was isolated from the B. napus T2 line and sequenced. Comparison of the two S regions revealed that (1) the gene organization downstream of SLG in both S haplotypes is highly colinear; (2) the distance between SLG-A14 and SRK-A14 genes is much larger than that between SLG-910 and SRK-910, with the intervening region filled with retroelements and haplotype-specific genes; and (3) the gene organization downstream of SRK in the two haplotypes is divergent. These observations lead us to propose that the SLG downstream region might be one border of the S locus and that the accumulation of heteromorphic sequences, such as retroelements as well as haplotype-unique genes, may act as a mechanism to suppress recombination between SLG and SRK.

Characterization and immunolocalization of a cytosolic calcium-binding protein from Brassica napus and Arabidopsis pollen.

Two low-molecular-weight proteins have been purified from Brassica napus pollen and a gene corresponding to one of them has been isolated. The gene encodes an 8.6-kD protein with two EF-hand calcium-binding motifs and is a member of a small gene family in B. napus. The protein is part of a family of pollen allergens recently identified in several evolutionarily distant dicot and monocot plants. Homologs have been detected in Arabidopsis, from which one gene has been cloned in this study, and in snapdragon (Antirrhinum majus), but not in tobacco (Nicotiana tabacum). Expression of the gene in B. napus was limited to male tissues and occurred during the pollen-maturation phase of anther development. Both the B. napus and Arabidopsis proteins interact with calcium, and the potential for a calcium-dependent conformational change was demonstrated. Given this affinity for calcium, the cloned genes were termed BPC1 and APC1 (B. napus and Arabidopsis pollen calcium-binding protein 1, respectively). Immunolocalization studies demonstrated that BPC1 is found in the cytosol of mature pollen. However, upon pollen hydration and germination, there is some apparent leakage of the protein to the pollen wall. BPC1 is also concentrated on or near the surface of the elongating pollen tube. The essential nature of calcium in pollen physiology, combined with the properties of BPC1 and its high evolutionary conservation suggests that this protein plays an important role in pollination by functioning as a calcium-sensitive signal molecule.

Molecular characterization of an Arabidopsis gene encoding hydroperoxide lyase, a cytochrome P-450 that is wound inducible.

Hydroperoxide lyase (HPL) cleaves lipid hydroperoxides to produce volatile flavor molecules and also potential signal molecules. We have characterized a gene from Arabidopsis that is homologous to a recently cloned HPL from green pepper (Capsicum annuum). The deduced protein sequence indicates that this gene encodes a cytochrome P-450 with a structure similar to that of allene oxide synthase. The gene was cloned into an expression vector and expressed in Escherichia coli to demonstrate HPL activity. Significant HPL activity was evident when 13S-hydroperoxy-9(Z),11(E),15(Z)-octadecatrienoic acid was used as the substrate, whereas activity with 13S-hydroperoxy-9(Z),11(E)-octadecadienoic acid was approximately 10-fold lower. Analysis of headspace volatiles by gas chromatography-mass spectrometry, after addition of the substrate to E. coli extracts expressing the protein, confirmed enzyme-activity data, since cis-3-hexenal was produced by the enzymatic activity of the encoded protein, whereas hexanal production was limited. Molecular characterization of this gene indicates that it is expressed at high levels in floral tissue and is wound inducible but, unlike allene oxide synthase, it is not induced by treatment with methyl jasmonate.

C6-volatiles derived from the lipoxygenase pathway induce a subset of defense-related genes.

Six-Carbon (C6-) volatiles, including the aldehydes trans-2-hexenal, hexanal and cis-3-hexenal, as well as their corresponding alcohols, are produced from damaged or wounded plant tissue as a product of the enzymatic activity of hydroperoxide lyase (HPL), a component of the lipoxygenase (LOX) pathway. Aerial treatment of Arabidopsis seedlings with 10 microM concentrations of trans-2-hexenal induces several genes known to be involved in the plant's defense response, including phenylpropanoid-related genes as well as genes of the LOX pathway. Genes encoding the pathogenesis-related proteins PR-1 or PR-2, however, were not induced. Trans-2-hexenal induction thus closely mimics the group of genes induced by methyl jasmonate (MeJA), also a LOX-derived volatile. However, unlike MeJA, trans-2-hexenal did not induce hydroxymethylglutaryl-coenzyme A reductase (HMGR) or thionin2-1. The inductive effect seemed to be limited to C6-related volatiles, as C8-, C9- and other related volatiles did not induce LOX mRNA levels. As has been demonstrated for MeJA, trans-2-hexenal quantitatively reduced wild-type seed germination. Trans-2-hexenal also reduced the germination frequency of the MeJA resistant Arabidopsis mutant, jar1-1, supporting the notion that trans-2-hexenal and MeJA are recognized via different mechanisms. In addition, trans-2-hexenal had a moderate inhibitory effect on root length relative to similar concentrations of MeJA and was approximately 10-fold less effective than MeJA at inducing anthocyanin accumulation in Arabidopsis seedlings. These results suggest that C6-volatiles of the LOX pathway act as a wound signal in plants, but result in a moderate plant response relative to MeJA at both the physiological and molecular level.

Footprinting of the spinach nitrite reductase gene promoter reveals the preservation of nitrate regulatory elements between fungi and higher plants.

Nitrite reductase (NiR) is the second enzyme in the nitrate assimilatory pathway reducing nitrite to ammonium. The expression of the NiR gene is induced upon the addition of nitrate. In an earlier study, a 130 bp upstream region of the spinach NiR gene promoter, located between -330 to - 200, was shown to be necessary for nitrate induction of beta-glucuronidase (GUS) expression in tissue-specific manner in transgenic tobacco plant [28]. To further delineate the cis-acting elements involved in nitrate regulation of NiR gene expression, transgenic tobacco plants were generated with 5' deletions in the -330 to -200 region of the spinach NiR gene promoter fused to the GUS gene. Plants with the NiR promoter deleted to -230 showed a considerable increase in GUS activity in the presence of nitrate, indicating that the 30 bp region between -230 to -200 is crucial for nitrate-regulated expression of NiR. In vivo DMS footprinting of the -300 to -130 region of the NiR promoter in leaf tissues from two independent transgenic lines revealed several nitrate-inducible footprints. Footprinting within the -230 to -181 region revealed factor binding to two adjacent GATA elements separated by 24 bp. This arrangement of GATA elements is analogous to cis-regulatory sequences found in the promoters of nitrate-inducible genes of Neurospora crassa, regulated by the NIT2 Zn-finger protein. The -240 to -110 fragment of the NiR promoter, which contains two NIT2 consensus core elements, bound in vitro to a fusion protein comprising the zinc finger domain of the N. crassa NIT2 protein. The data presented here show that nitrate-inducible expression of the NiR gene is mediated by nitrate-specific binding of trans-acting factors to sequences preserved between fungi and higher plants.

Developmental regulation of two tomato lipoxygenase promoters in transgenic tobacco and tomato.

Two lipoxygenase (LOX) genes (tomloxA and tomloxB) are expressed in ripening tomato fruit, and tomloxA is also expressed in germinating seedlings. The 5'-upstream regions of these genes were isolated to study the regulatory elements involved in coordinating tomlox gene expression. Sequence analysis of the promoters did not reveal any previously characterized regulatory elements except for TATA and CAAT boxes. However, the sequence motif GATAcAnnAAtnTGATG was found in both promoters. Chimeric gene fusions of each tomlox promoter with the beta-glucuronidase reporter gene (gus) were introduced into tobacco and tomato plants via Agrobacterium-mediated transformation. GUS activity in tomloxA-gus plants during seed germination peaked at day 5 and was enhanced by methyl jasmonate (MeJa) treatment. No GUS activity was detected in tomloxB-gus seedlings. Neither wounding nor abscisic acid (ABA) treatment of transgenic seedlings modified the activity of either promoter. During fruit development, GUS expression in tomloxA-gus tobacco fruit increased 5 days after anthesis (DAA) and peaked at 20 DAA. In tomloxB-gus tobacco fruit, GUS activity increased at 10 DAA and peaked at 20 DAA. In transgenic tomato fruit, tomloxA-gus expression was localized to the outer pericarp during fruit ripening, while tomloxB-gus expression was localized in the outer pericarp and columella. These data demonstrate that the promoter regions used in these experiments contain cis-acting regulatory elements required for proper regulation of tomlox expression during development and for MeJa-responsiveness.

Molecular characterization of the S locus in two self-incompatible Brassica napus lines.

In Brassica species, self-incompatibility has been mapped genetically to a single chromosomal location. In this region, there are two closely linked genes coding for the S locus glycoprotein (SLG) and S locus receptor kinase (SRK). They appear to comprise the pistil component of the self-incompatibility reaction. SLG and SRK are thought to recognize an unknown pollen component on the incompatible pollen, and the gene encoding this pollen component must also be linked to the SLG and SRK genes. To further our understanding of self-incompatibility, the chromosomal region carrying the SLG and SRK genes has been studied. The physical region between the SLG-910 and the SRK-910 genes in the Brassica napus W1 line was cloned, and a search for genes expressed in the anther revealed two additional S locus genes located downstream of the SLG-910 gene. Because these two genes are novel and are conserved at other S alleles, we designated them as SLL1 and SLL2 (for S locus-linked genes 1 and 2, respectively). The SLL1 gene is S locus specific, whereas the SLL2 gene is not only present at the S locus but is also present in other parts of the genomes in both self-incompatible and self-compatible Brassica ssp lines. Expression of the SLL1 gene is only detectable in anthers of self-incompatible plants and is developmentally regulated during anther development, whereas the SLL2 gene is expressed in anthers and stigmas in both self-incompatible and self-compatible plants, with the highest levels of expression occurring in the stigmas. Although SLL1 and SLL2 are linked to the S locus region, it is not clear whether these genes function in self-incompatibility or serve some other cellular roles in pollen-pistil functions.

Two members of the thioredoxin-h family interact with the kinase domain of a Brassica S locus receptor kinase.

To determine potential targets of the S locus receptor kinase (SRK) during the Brassica self-incompatibility response, a yeast two-hybrid library was screened with the SRK-910 protein kinase domain. Two thioredoxin-h-like clones, THL-1 and THL-2, were found to interact specifically with the SRK-910 protein kinase domain and not to interact with the protein kinase domains from the Arabidopsis receptor-like protein kinases (RLK) RLK4 and RLK5. The interaction between THL-1 and the SRK-910 protein kinase domain was confirmed using coimmunoprecipitation experiments with fusion proteins produced in Escherichia coli. THL-1 has thioredoxin activity based on an insulin reduction assay, and THL-1 is weakly phosphorylated by the SRK-910 protein kinase domain. THL-1 and THL-2 are both expressed in a variety of tissues but show some differences in steady state mRNA levels, with THL-2 being preferentially expressed in floral tissues. This indicates a more general biological function for these thioredoxins in addition to a potential role as effector molecules in the self-incompatibility signal cascade.

Engineering resistance to trypsin inactivation into L-asparaginase through the production of a chimeric protein between the enzyme and a protective single-chain antibody.

We have demonstrated that a trypsin sensitive enzyme such as L-asparaginase can be rendered trypsin resistant by genetically fusing its gene with that of a single-chain antibody derived from a preselected monoclonal antibody capable of providing protection against trypsin. The chimeric L-asparaginase retained 75% of its original activity upon exposure to trypsin, whereas the native unprotected L-asparaginase control was totally inactivated.

The cloning of two tomato lipoxygenase genes and their differential expression during fruit ripening.

A membrane-associated lipoxygenase from breaker-stage fruit of tomato (Lycopersicon esculentum Mill.) was purified and partially sequenced. Using degenerate oligonucleotides corresponding to portions of this sequence, a cDNA was amplified by PCR and used to screen a breaker fruit cDNA library. Two clones, tomloxA and tomloxB, were isolated and one of these (tomloxA) corresponded to the isolated protein. Genomic clones were isolated and sequence data from these were used to obtain the 5' ends of the cDNAs. The 2.8-kb cDNAs encode proteins that are similar in size and sequence to each other and to other plant lipoxygenases. DNA blot analysis indicated that tomato contains three or more genes that encode lipoxygenase. RNA blot analysis showed that tomloxA is expressed in germinating seeds as well as in ripening fruit, where it reached its peak during breaker stage. tomloxB appears to be fruit specific and is at its highest level in ripe fruit.

Features of the extracellular domain of the S-locus receptor kinase from Brassica.

An S-receptor kinase (SRK) gene associated with self-incompatibility in a Brassica napus subsp. oleifera line has been characterized. The SRK-A14 cDNA shows the highest levels of homology in the 5' end to the SLG-A14 cDNA present at the same locus. RNA blot analysis shows that the SRK-A14 gene is expressed predominantly in the pistil, and at lower levels in the anthers. The predicted amino acid sequences from the extracellular domain of the SRK-A14 gene and three other SRK genes were compared. The different SRK extracellular domains were for the most part very similar, with the exception of two variable regions containing a high level of amino acid alterations. These extracellular domains also contain a region of similarity to the immunoglobulin domains present in members of the immunoglobulin superfamily. These findings may define regions of the SRK protein that are necessary for interactions between SRK and other proteins.